Here we analyse the metabolite data from the maize root bacteria screening experiment in December 2019.

Some strains had to be excluded because they grew bad in the assay. This was checked by optical density measurement of the 96 well plates (which was prone to give false results because there condensed water forms below the lid which affects the measurements) and by measuring two amino acids in the cultures

Strain Genus Phylum Colour_Met
LRH8.O Rhizobium Proteobacteria NA
LST17 Stenotrophomonas Proteobacteria NA
LMX3 Micrococcineae Actinobacteria NA
LMX8 Bacillus Firmicutes NA
LMC1 Pantoea Proteobacteria NA
LMX11 Pseudomonas Proteobacteria NA
LMK1 Pantoea Proteobacteria NA
LME2 Bacillus Firmicutes NA

Dilutions

All the samples (except T0 samples) were diluted with 100 % MeOH in a ratio (30:70 - sample:MeOH). Then they were diluted 1:6 in the pooled eppi and 1:10 in the analysis vial, resulting in a final dilution of 200. The T0 samples were diluted 1:6. Therefore the values have to be adjusted to the dilution.

BX profiles of media controls

Benzoxazinoids were mixed to TSB growth media, each at a concentration of 500 uM. BXs were MBOA, DIMBOA and DIMBOA-Glc. The treatments were diluted and frozen directly at the start of the experiment (T0) and incubated without bacteria throughout the experiment (NBC). The T0 samples were differently diluted at the beginning, therefore they were here normalized with MBOA values (*35) - MBOA is stable in these conditions. NBC and T0 plotted show the spontaneous degradation of the BXs in the culture growth conditions.

MBOA seems to be pure and stable over the course of the experiment. DIMBOA is not stable and degrades completly to MBOA. As the profile shows at T0 it is not pure and also contains HMBOA which degrades to BOA.

DIMBOA-Glc is stable but not pure.

All metabolites in the cultures

Quantitative analysis

Bargraphs

Heatmap

Genus information

Fraction compound metabolized

As MBOA and DIMBOA are quite stable in the growth conditions in liquid cultures, the fraction of metabolized compound can be easily calculated for each strain. The value for the certain compound (uMol) in a certain strain is substracted from the NBC treatment & divided by the NBC treatment value and multiplyied by 100 to get percentage.

Qualitative analysis

Since there is quite some variation in the absolute values of metabolites in the cultures, which makes it difficult to categorize the bacteria to metabolic types, we to qualitative categorization of the bacteria to groups. We define threshold concentrations of the supplemented compounds (MBOA & DIMBOA-Glc) to group bacteria into “degraders” or “non-degraders” and for metabolization products (AMPO & AAMPO) to group in “formers” and “non-formers”.

MBOA degradation We define MBOA degradation with a threshold of 70 % MBOA compared to NBC detected (587.5834 μM). This corresponds to 411.3084 and we set the threshold to 400 μM.

AMPO formation > 0.13 μM

Weak AMPO formation: To define the threshold for AMPO formation, we check the amount of AMPO produced by the Rhizobia which show a weak colour change in the plate assay, consistently form little AMPO, and degrades MBOA. 0.13 μM - 10 μM

Strong AMPO formation: To define the threshold for strong AMPO formation, we check the amount of AMPO formed by Pseudoarthrobacter which consistently shows an intensive red colour on the plate assay, red precipitation in liquid medium and degrades MBOA. > 10 μM

AAMPO formation Same as for AMPO > 0.13 μM

DIMBOA-Glc degradation We define DIMBOA-Glc degradation with a threshold of 70 % DIMBOA-Glc compared to NBC detected (246.9373 μM). This value is much lower compared to MBOA because the DIMBOA-Glc we used was only of 70% purity. This corresponds to 172.8561 and we set the threshold to 170 μM.

MBOA formation from DIMBOA-Glc

We define MBOA formation from DIMBOA-Glc with a threshold of 10 % MBOA compared to the maximum of MBOA detected in DIMBOA-Glc treated samples (143.2905 μM). We set the threshold to 14 μM.

Qualitative Graphs

Summary table

Chem No_met Met MBOA AMPO AAMPO unknown total
MBOA 30 15 NA 13 4 2 45
DMG 25 13 0 9 3 8 38

Venn Diagramms metabolism

Comparing metabolism patterns across strains with a VennDiagramm.

## $q1
## character(0)
## 
## $q2
## [1] "LRH8.S" "LRC7.O" "LRH13"  "LRC7.S" "LMD1"   "LME3"   "LMA1"   "LAR21" 
## 
## $q3
## named list()
## 
## $q4
## character(0)
## 
## $q5
## [1] "LMX7"  "LAC11" "LMC3"  "LSP13" "LMB2" 
## 
## $q6
## $q6$Strain
##  [1] "LMG1"   "LMX7"   "LST16"  "LST14"  "LBA3"   "LST13"  "LAC11"  "LST12" 
##  [9] "LBA112" "LML1"   "LBA71"  "LMH1"   "LST11"  "LME3"   "LMA1"   "LAR21" 
## [17] "LMB2"  
## 
## $q6$uMol
##  [1] 0.1755502 1.7599770 0.2159358 0.1985998 0.2230228 0.2358932 0.3901446
##  [8] 0.2620072 0.1816390 0.1711316 0.2202242 0.1940634 3.8101014 4.2116710
## [15] 0.1648716 0.8374710 0.9274850
## 
## 
## $q7
## [1] "LRH11"
## character(0)
## character(0)
## character(0)
## character(0)
## R version 4.3.1 (2023-06-16)
## Platform: x86_64-apple-darwin20 (64-bit)
## Running under: macOS Ventura 13.3.1
## 
## Matrix products: default
## BLAS:   /Library/Frameworks/R.framework/Versions/4.3-x86_64/Resources/lib/libRblas.0.dylib 
## LAPACK: /Library/Frameworks/R.framework/Versions/4.3-x86_64/Resources/lib/libRlapack.dylib;  LAPACK version 3.11.0
## 
## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
## 
## time zone: Europe/Zurich
## tzcode source: internal
## 
## attached base packages:
## [1] grid      stats     graphics  grDevices utils     datasets  methods  
## [8] base     
## 
## other attached packages:
##  [1] reshape2_1.4.4     ggdendro_0.1.23    pheatmap_1.0.12    RColorBrewer_1.1-3
##  [5] tibble_3.2.1       pander_0.6.5       ggpmisc_0.5.4-1    ggpp_0.5.4        
##  [9] forcats_1.0.0      stringr_1.5.0      readr_2.1.4        tidyr_1.3.0       
## [13] readxl_1.4.2       ggplot2_3.4.3      magrittr_2.0.3     dplyr_1.1.2       
## 
## loaded via a namespace (and not attached):
##  [1] sass_0.4.6         utf8_1.2.3         generics_0.1.3     stringi_1.7.12    
##  [5] lattice_0.21-8     hms_1.1.3          digest_0.6.31      evaluate_0.21     
##  [9] fastmap_1.1.1      plyr_1.8.8         cellranger_1.1.0   jsonlite_1.8.5    
## [13] Matrix_1.5-4.1     survival_3.5-5     purrr_1.0.1        fansi_1.0.4       
## [17] viridisLite_0.4.2  scales_1.2.1       jquerylib_0.1.4    cli_3.6.1         
## [21] rlang_1.1.1        splines_4.3.1      munsell_0.5.0      withr_2.5.0       
## [25] cachem_1.0.8       yaml_2.3.7         tools_4.3.1        MatrixModels_0.5-1
## [29] SparseM_1.81       polynom_1.4-1      tzdb_0.4.0         colorspace_2.1-0  
## [33] vctrs_0.6.3        R6_2.5.1           lifecycle_1.0.3    MASS_7.3-60       
## [37] pkgconfig_2.0.3    pillar_1.9.0       bslib_0.5.0        gtable_0.3.4      
## [41] Rcpp_1.0.11        glue_1.6.2         highr_0.10         xfun_0.39         
## [45] tidyselect_1.2.0   rstudioapi_0.14    knitr_1.43         farver_2.1.1      
## [49] htmltools_0.5.5    labeling_0.4.2     rmarkdown_2.22     compiler_4.3.1    
## [53] quantreg_5.95